CN110078619B - Cyclotetatetranol ester compound extracted, separated and purified from green bamboo marks and method thereof - Google Patents

Cyclotetatetranol ester compound extracted, separated and purified from green bamboo marks and method thereof Download PDF

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CN110078619B
CN110078619B CN201910407238.9A CN201910407238A CN110078619B CN 110078619 B CN110078619 B CN 110078619B CN 201910407238 A CN201910407238 A CN 201910407238A CN 110078619 B CN110078619 B CN 110078619B
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CN110078619A (en
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于金倩
王晓
耿岩玲
张敏敏
王岱杰
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Shandong Analysis and Test Center
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Abstract

The invention relates to a cyclotetanolate compound extracted, separated and purified from a green bamboo label and a method thereof. Has a structure shown in a formula I,
Figure DDA0002061636430000011
wherein R is1Is selected from
Figure DDA0002061636430000012
Or
Figure DDA0002061636430000013
R2Is selected from
Figure DDA0002061636430000014
Or a hydrogen radical; r3Selected from hydroxy or
Figure DDA0002061636430000015
R4Selected from hydrogen radicals or hydroxyl radicals. Has better antioxidant activity and good medicinal prospect.

Description

Cyclotetatetranol ester compound extracted, separated and purified from green bamboo marks and method thereof
Technical Field
The invention belongs to the technical field of cyclotetanol ester compounds and preparation methods thereof, and particularly relates to a cyclotetanol ester compound extracted, separated and purified from green bamboo labels and a method thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The green bamboo label (Scindapsus of fiscinalis Schott) is named as Millettia odorata, Millettia speciosa, climbing tree dragon, golden bamboo label, and the whole plant of the Araceae Marsdenia tenacissima, mainly distributed in Yunnan, Guizhou and Guangxi, and is a rare ethnic medicine widely applied in local places. Recorded in Yunnan Chinese herbal medicine selection, the medicine has the effects of removing blood stasis, relieving pain, moistening lung and relieving cough, and can be used for treating traumatic injury, fracture, rheumatic numbness, bronchitis and pertussis; recorded in Guangxi medicine plant famous book, the medicine has the effects of relieving swelling and pain, and treating traumatic injury, rheumatism and carbuncle sore; the book of Guizhou medicine plant records that the medicine can remove blood stasis, promote tissue regeneration and relieve pain. The green bamboo mark has better research prospect. The cyclotetriptan ester compounds have not been reported to be extracted from other plant species. In the prior art, chromone ketoside compounds and alkaloid compounds are extracted from the green bamboo labels, and the green bamboo labels are used for lung clearing medicines, scald medicines, rhinitis, spinal cord injury, fracture, health-care products for strengthening spleen and stomach, and the like.
Disclosure of Invention
In view of the problems in the prior art, it is an object of the present invention to provide a cyclotetanols compound extracted, separated and purified from the green bamboo culm and a method thereof.
In order to solve the technical problems, the technical scheme of the invention is as follows:
on the first hand, the cyclotetanolate compounds extracted, separated and purified from the green bamboo labels have the structure shown in the formula I,
Figure GDA0003308701230000011
wherein R is1Is selected from
Figure GDA0003308701230000012
R2Is selected from
Figure GDA0003308701230000013
Or a hydrogen radical; r3Selected from hydroxy or
Figure GDA0003308701230000021
R4Selected from hydrogen radicals or hydroxyl radicals.
In some embodiments, the above-mentioned cyclotetetranol ester compound has the structure shown in formula II and formula III,
Figure GDA0003308701230000022
wherein n is1Is taken from 0 or 1; n is2Is taken from 0 or 1; r5Selected from hydroxy or
Figure GDA0003308701230000023
R7Is selected from
Figure GDA0003308701230000024
Or a hydroxyl group; r6Is selected from
Figure GDA0003308701230000025
R8Selected from hydrogen radicals or hydroxyl radicals. In still other embodiments, the compound of formula II is selected from the group consisting of1When 0, n2Is 1, R5Is composed of
Figure GDA0003308701230000026
Obtaining the compound shown in the formula (IV),
Figure GDA0003308701230000031
in still other embodiments, the compound of formula II is selected from the group consisting of1When 1, n2Is 0, R5Is hydroxyl to obtain the compound shown in the formula (V),
Figure GDA0003308701230000032
in still other embodiments, the compound of formula III is selected from the group consisting of7When it is hydroxy, R8When it is hydroxy, R6Is selected from
Figure GDA0003308701230000033
Figure GDA0003308701230000034
To obtain the compound shown in the formula (VI) or the formula (VII),
Figure GDA0003308701230000035
in still other embodiments, the compound of formula III is selected from the group consisting of7When it is hydroxy, R8When it is hydrogen, R6Is composed of
Figure GDA0003308701230000036
To obtain the compound shown in the formula (VIII),
Figure GDA0003308701230000037
in still other embodiments, the compound of formula III is selected from the group consisting of4Is composed of
Figure GDA0003308701230000038
When R is5Is composed of
Figure GDA0003308701230000039
To obtain the compound shown as the formula (IX),
Figure GDA00033087012300000310
in a second aspect, the method for extracting the cyclotetetraol ester compound comprises the following specific steps:
performing crude extraction on the green bamboo label, sequentially extracting the crude extract by using petroleum ether and ethyl acetate, and removing a solvent from an extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
the ethyl acetate extract was dissolved in CH2Cl2-MeOH, gradient elution with silica gel chromatography, gradient elution of CH2Cl2-the volume ratio of MeOH, in the order 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1, dividing the eluent of each gradient into more than 8 parts of receiving solution;
combining and concentrating the receiving solutions of the set sections according to the receiving sequence, eluting and separating the concentrate through a C18 adsorption resin chromatographic column, wherein gradient eluents of the C18 adsorption resin chromatographic column are 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH in sequence, and dividing each gradient eluent into more than 8 parts of receiving solutions;
taking the receiving liquid of the set section according to the receiving sequence, using CH3CN and H2And eluting and separating the mixed solution of O to obtain the compound shown in the formula (I).
The inventor finds that when the eluent of gradient elution is separated and the quantity of the receiving volume is in a certain range, the probability of separating other components with different polarities is greatly improved, and then CH is used for extracting the eluent3CN and H2Elution with a mixed solution of O, CH3CN and H2The mixed solution of O has a larger polarity, but a polarity smaller than that of water, so that the compound is separated from the eluent to obtain the final target product.
CH2Cl2MeOH elutes because the polarity of the mixture of the two is suitable for the separation of the ethyl acetate extract.
In some embodiments, each gradient of eluent is divided into 8, 9, or 10 portions of receiving solution; in still other embodiments, the volume of eluent for each gradient is 4000-10000mL, and each 400-1000mL is a receiving volume; further, the volume eluted per gradient was 5000mL, one receiving volume per 500 mL.
It is understood that when the volume of the eluent for each gradient is 5000mL and one receiving volume is 500mL, then one receiving volume is 500mL, and one skilled in the art can select several receiving volumes according to the detected compound.
In some embodiments, the first silica gel column is subjected to gradient elution in which the first 5 receiving solutions in a 15:1 gradient are combined and concentrated to provide concentrate A, which is dissolved in CH2Cl2-MeOH in a volume ratio of 15:1, and gradient elution with CH by silica gel chromatography2Cl2-MeOH at a volumetric ratio of 100:1 → 50:1 → 25:1 → 15:1, dividing the eluate from each gradient into more than 8 portions of receiver, and combining the resulting 15:1 gradients of receivers to give an eluted fraction B;
elution fraction B through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and dividing each gradient eluent into more than 8 parts of receiving solution;
the 5 th receiver of the elution fraction of a 30. + -. 1% MeOH gradient was treated with CH3CN and H2And eluting and separating the mixed solution of O as a mobile phase to obtain the compound shown in the formula (VI).
High Performance Liquid Chromatography (HPLC) analysis revealed that the compound of formula (VI) was predominantly present in the 5 th receiving volume of the eluate.
Preferably, CH3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 15: 80-90.
Preferably, elution is with MeOH, which has a flow rate of 45-55 mL/min.
Preferably, with CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
When the extractant is used for extraction, the polarities and chemical bonds of all compounds in the cyclotetetranol ester compounds are different, and elution parts, different receiving volumes and CH are obtained by different gradients3CN and H2The proportion of O, directly affecting the type of compound obtained, since the contents and compositions of the components obtained in the individual receiving volumes after separation are also different, the inventors have found that the operating parameters have a great influence on the components and compositions in the obtained receiving volumes.
In some embodiments, in the first silica gel column gradient elution, the first 5 receiving solutions in a 15:1 gradient are combined and concentrated to provide concentrate C, which is dissolved in CH2Cl2-MeOH in a volume ratio of 15:1, and gradient elution with CH by silica gel chromatography2Cl2-MeOH at a volumetric ratio of 100:1 → 50:1 → 25:1 → 15:1, dividing each gradient into more than 8 portions of receiving fluid, and combining the 15:1 gradients of receiving fluid to provide an eluted fraction D;
elution fraction D through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and taking the eluent of each gradient as more than 8 parts of receiving solution;
the 3 rd receiver of the elution fraction with a 45. + -. 1% MeOH gradient was applied with CH3CN and H2And (5) taking the mixed solution of O as a mobile phase for elution and separation to obtain the compound shown in the formula (VII).
Preferably, CH3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 20: 75-95.
Preferably, elution is with MeOH, which has a flow rate of 45-55 mL/min.
Preferably, with CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
In some embodiments, the first silica gel column is subjected to gradient elution in which the last 5 receiving solutions in a 15:1 gradient are concentrated to provide concentrate E, which is dissolved in CH2Cl2-MeOH in a volume ratio of 15:1, and gradient elution with CH by silica gel chromatography2Cl2MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1, dividing the eluent of each gradient into large fractionsCombining the receiving solutions in a gradient of 15:1 in 8 parts of the receiving solution to obtain an elution part F;
elution fraction F through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and dividing each gradient eluent into more than 8 parts of receiving solution;
the 2 nd receiver of the elution fraction of a 30. + -. 1% MeOH gradient was treated with CH3CN and H2And (4) taking the mixed solution of O as a mobile phase for elution separation to obtain the compound shown in the formula (V).
Preferably, CH3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 14: 80-90.
Preferably, elution is with MeOH, which has a flow rate of 45-55 mL/min.
Preferably, with CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
In some embodiments, in the first silica gel column gradient elution, the last five receiving solutions of the 10:1 gradient are combined and concentrated to give concentrate G; passing the condensate G through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and dividing each gradient eluent into more than 8 parts of receiving solution; passing the second 2 receiving solutions of the 45 +/-1% MeOH gradient elution part through a Sepadex LH-20 gel resin chromatographic column, and eluting with MeOH, wherein the eluent is divided into more than 8 receiving solutions;
elution fraction 3 with MeOH site CH3CN and H2And eluting and separating the mixed solution of O as a mobile phase to obtain the compound shown in the formula (VIII).
MeOH is anhydrous methanol.
Preferably, CH3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 20: 75-85.
Preferably, elution is with MeOH, which has a flow rate of 45-55 mL/min.
Preferably, with CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
In some embodiments, in the first silica gel column gradient elution, the first 2 accepts of the 5:1 gradient are combined and concentrated to provide concentrate H; passing concentrate H through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and dividing each gradient eluent into more than 8 parts of receiving solution; passing the second 2 receiving solutions of the 45 +/-1% MeOH gradient elution part through a Sepadex LH-20 gel resin chromatographic column, and eluting with MeOH, wherein the eluent is divided into more than 8 receiving solutions;
elution fraction 6 with MeOH site CH3CN and H2And (4) eluting and separating the mixed solution of O as a mobile phase to obtain the compound shown in the formula (IV).
The 6 th elution site corresponds to the 6 th receiving solution.
Preferably, CH3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 28: 70-75.
Preferably, elution is with MeOH, which has a flow rate of 45-55 mL/min.
Preferably, with CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
In some embodiments, in the first silica gel column gradient elution, the first 2 accepts of the 5:1 gradient are combined and concentrated to give concentrate K; sequentially using 12 + -1% MeOH, 30 + -1% MeOH, 45 + -1% MeOH, 60 + -1% MeOH18Eluting by using an adsorption resin chromatographic column, and dividing each gradient eluent into more than 8 parts of receiving solution; passing the first 1 receiving volumes of the second part of the 45 +/-1% MeOH gradient elution part through a Sepadex LH-20 gel resin chromatographic column, eluting with MeOH, and dividing the eluent into more than 8 parts of receiving solution;
elution fraction 8 with MeOH with CH3CN and H2Eluting and separating the mixed solution of O as a mobile phase to obtain the compound shown as the formula (IX)。
Preferably, CH3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 28: 70-75.
Preferably, elution is with MeOH, which has a flow rate of 45-55 mL/min.
Preferably, with CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
In a third aspect, the use of the cyclotetetranol ester compound in the preparation of an anti-oxidation medicament.
The invention has the beneficial effects that:
the new cyclotetritol ester compound separated and purified from the green bamboo marks and the preparation method thereof take the green bamboo marks as raw materials, the sources are wide, the preparation process is simple, economic and safe, the yield is high, the compounds in 6 obtained new compounds all have certain antioxidant activity, and the compound VI has better antioxidant activity and good medicinal prospect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the invention and not to limit the invention.
FIG. 1 is a compound of formula (IV)1H NMR；
FIG. 2 is a drawing of a compound of formula (IV)13C NMR；
FIG. 3 is a compound of formula (IX)1H NMR；
FIG. 4 is a compound of formula (IX)13C NMR；
FIG. 5 is a drawing of a compound of formula (V)1H NMR；
FIG. 6 is a drawing of a compound of formula (V)13C NMR；
FIG. 7 is a drawing showing a compound of formula (VI)1H NMR；
FIG. 8 is a drawing of a compound of formula (VI)13C NMR；
FIG. 9 is a drawing of a compound of formula (VII)1H NMR；
FIG. 10 is a drawing of a compound of formula (VII)13C NMR；
FIG. 11 is a drawing of a compound of formula (VIII)1H NMR；
FIG. 12 is a drawing of a compound of formula (VIII)13C NMR。
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The structural formula of the compound IV is
Figure GDA0003308701230000071
Is named as (2E,2' E) - (1R,2R,3S,4S) -3,4-dihydroxy-3- (3-methoxy-3-oxypropy) cyclobutane-1,2-diylbis [3- (3,4-dihydroxyphenyl) acrylate]；
The compound IX has a structural formula
Figure GDA0003308701230000081
Is named as (2E,2' E) - (1S,2S,3R,4R) -2,4-dihydroxy-2- (3-methoxy-3-oxypropyl) cyclobutane-1,3-diylbis [3- (3,4-dihydroxyphenyl) acrylate]；
The structural formula of the compound V is
Figure GDA0003308701230000082
Is named as (E) - (1R,2R,3S,4S) -2,3,4-trihydroxy-3- (3-methoxy-3-oxypropyl) cyclobutylyl-3- (3,4-dihydroxyphenyl) acrylate;
compound VIThe structural formula of the compound is shown as the following formula,
Figure GDA0003308701230000083
is named as (E) - (1S,2S,3R,4R) -2,3,4-trihydroxy-2- (3-methoxy-3-oxypropyl) cyclobutyl3- (3,4-dihydroxyphenyl) acrylate;
the structural formula of the compound VII is
Figure GDA0003308701230000084
Is named as (E) - (1S,2R,3R,4S) -2- (3-ethoxy-3-oxopropyl) -2,3, 4-trihydroxybutyryl 3- (3,4-dihydroxyphenyl) acrylate;
the structural formula of the compound VIII is
Figure GDA0003308701230000085
Is named as (E) - (1S,2R,3R,4S) -2,3,4-trihydroxy-2- (3-methoxy-3-oxypropy) cyclobutyl3- (4-hydroxypentyl) acrylate. The invention will be further illustrated by the following examples
The apparatus used in the following examples was a semi-preparative high performance liquid chromatograph.
Example 1
A method for preparing a compound shown as a formula (VI).
(1) Taking 5kg of the green bamboo standard medicinal material, crushing, heating and refluxing with 95% ethanol at a solid-liquid ratio of 1:3 for three times of 2h, 1h and 1h respectively, combining the filtrates, carrying out reduced pressure rotary steaming, and freeze-drying to obtain 1kg of the green bamboo standard crude extract;
(2) adding appropriate amount of water into the crude extract, ultrasonic pulverizing, extracting with petroleum ether, ethyl acetate and n-butanol, filtering the extractive solution, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate, n-butanol and water extract;
(3) dissolving 180g of the obtained ethyl acetate part in methanol, adding 300g of 200-300-mesh silica gel for sample mixing, volatilizing the solvent, and performing volume ratio CH2Cl2Gradient elution with MeOH (100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 1:1 → 0:1) to give elution sites, concentration, 5000mL per gradient wash, one receiving volume per 500mL of receiving solution.
(4) Will CH2Cl2-MeOH (15:1) gradient elution site first 5 receiver pooled and concentrated, concentrate dissolved in CH2Cl2Adding silica gel filler into MeOH (15:1), stirring, evaporating to remove solvent, and gradient eluting with CH by silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1, gradient elution 2500mL, receiving volume of 1 receiving solution per 250 mL; subjecting the second silica gel column chromatography CH2Cl2The fraction eluting in MeOH (15:1) is passed through C18Adsorbing resin chromatographic column, eluting with 12% MeOH, 30% MeOH, 45% MeOH, and 60% MeOH sequentially, wherein each gradient elutes 2500mL, each 500mL is 1 receiving volume of receiving solution, and the flow rate is 50 mL/min; c is to be18Column chromatography 30% MeOH elution portion of the 5 th receiving solution CH3CN and H2And preparing a mobile phase with the volume ratio of O being 15:85, wherein the flow rate is 3mL/min, the detection wavelength is 310nm, and 50 mu L of the compound VI is obtained by injecting each sample.
Example 2
A method for preparing a compound shown as a formula (VII).
(1) Taking 5kg of the green bamboo standard medicinal material, crushing, heating and refluxing with 95% ethanol at a solid-liquid ratio of 1:3 for three times of 2h, 1h and 1h respectively, combining the filtrates, carrying out reduced pressure rotary steaming, and freeze-drying to obtain 1kg of the green bamboo standard crude extract;
(2) adding appropriate amount of water into the crude extract, ultrasonic pulverizing, extracting with petroleum ether, ethyl acetate and n-butanol, filtering the extractive solution, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate, n-butanol and water extract;
(3) dissolving 180g of the obtained ethyl acetate part in methanol, adding 300g of 200-300-mesh silica gel for sample mixing, volatilizing the solvent, and performing volume ratio CH2Cl2Gradient elution with MeOH (100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 1:1 → 0:1) to give elution sites, concentration, washing 5000mL per gradient, one receiving volume per 500mL of receiving solution.
(4) Will CH2Cl2-MeOH (15:1) gradient elution site first 5 receiver pooled and concentrated, concentrate dissolved in CH2Cl2Adding silica gel filler into MeOH (15:1), stirring, evaporating to remove solvent, and gradient eluting with CH by silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1, gradient elution 2500ml, receiving volume of 1 receiving solution per 250 ml; subjecting the second silica gel column chromatography CH2Cl2The fraction eluting in MeOH (15:1) is passed through C18Adsorbing resin chromatographic column, eluting with 12% MeOH, 30% MeOH, 45% MeOH, and 60% MeOH sequentially, wherein each gradient elutes 2500mL, each 500mL is 1 receiving volume of receiving solution, and the flow rate is 50 mL/min; c is to be18Column chromatography 45% MeOH elution portion of the 3 rd receiving solution CH3CN and H2Preparing a mobile phase with the volume ratio of O being 20:80, wherein the flow rate is 3mL/min, the detection wavelength is 310nm, and 50 mu L of sample is injected each time to obtain 7mg of a compound VII;
example 3
A preparation method of a compound shown as a formula (V).
(1) Taking 5kg of the green bamboo standard medicinal material, crushing, heating and refluxing with 95% ethanol at a solid-liquid ratio of 1:3 for three times of 2h, 1h and 1h respectively, combining the filtrates, carrying out reduced pressure rotary steaming, and freeze-drying to obtain 1kg of the green bamboo standard crude extract;
(2) adding appropriate amount of water into the crude extract, ultrasonic pulverizing, extracting with petroleum ether, ethyl acetate and n-butanol, filtering the extractive solution, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate, n-butanol and water extract;
(3) dissolving 180g of the obtained ethyl acetate part in methanol, adding 300g of 200-300-mesh silica gel for sample mixing, volatilizing the solvent, and performing volume ratio CH2Cl2Gradient elution with MeOH (100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 1:1 → 0:1) to give elution sites, concentration, washing 5000mL per gradient, one receiving volume per 500mL of receiving solution.
(4) Subjecting the first silica gel column chromatography CH2Cl2-MeOH (15:1) gradient elution site last 5 receiving liquid and concentrating, the concentrate is dissolved in CH2Cl2Adding silica gel filler into MeOH (15:1), stirring, volatilizing solvent, and purifying by silica gel chromatographic columnGradient elution with CH2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1, gradient elution 2500ml, receiving volume of 1 receiving solution per 250 ml; subjecting the second silica gel column chromatography CH2Cl2The fraction eluting in MeOH (15:1) is passed through C18Adsorbing resin chromatographic column, eluting with 12% MeOH, 30% MeOH, 45% MeOH, and 60% MeOH sequentially, wherein each gradient elutes 2500mL, each 500mL is 1 receiving solution, and the flow rate is 50 mL/min; c is to be18Column chromatography 30% MeOH elution portion 2 nd receiving liquid with CH3CN and H2And preparing a mobile phase with the volume ratio of O being 14:86, wherein the flow rate is 3mL/min, the detection wavelength is 310nm, and 50 mu L of the compound V is obtained by injection for each time.
Example 4
A method for preparing a compound shown as a formula (VIII).
(1) Taking 5kg of the green bamboo standard medicinal material, crushing, heating and refluxing with 95% ethanol at a solid-liquid ratio of 1:3 for three times of 2h, 1h and 1h respectively, combining the filtrates, carrying out reduced pressure rotary steaming, and freeze-drying to obtain 1kg of the green bamboo standard crude extract;
(2) adding appropriate amount of water into the crude extract, ultrasonic pulverizing, extracting with petroleum ether, ethyl acetate and n-butanol, filtering the extractive solution, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate, n-butanol and water extract;
(3) dissolving 180g of the obtained ethyl acetate part in methanol, adding 300g of 200-300-mesh silica gel for sample mixing, volatilizing the solvent, and performing volume ratio CH2Cl2Gradient elution with MeOH (100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 1:1 → 0:1) to give elution sites, concentration, 5000mL per gradient wash, one receiving volume per 500mL of receiving solution.
(4) Subjecting the first silica gel column chromatography CH2Cl2The last 5 recipients of the MeOH (10:1) gradient elution site were combined and concentrated by C18Performing resin adsorption chromatography, and sequentially eluting with 12% MeOH, 30% MeOH, 45% MeOH, and 60% MeOH, wherein each gradient elutes 2500mL, each 500mL is 1 receiving solution, and the flow rate is 50 mL/min; c is to be18Column chromatography 45% MeOH elution siteThe next 2 receiving volumes pass through a Sephadex LH-20 gel resin chromatographic column, and are eluted by MeOH, and each 10mL of the receiving volumes is 1 receiving solution; eluting the 3 rd elution part of MeOH in the SephadexLH-20 gel column chromatography with CH3CN and H2And preparing a mobile phase with the volume ratio of O being 20:80, wherein the flow rate is 3mL/min, the detection wavelength is 310nm, and 50 mu L of the compound VIII is obtained by feeding each sample.
Example 5
A preparation method of a compound shown as a formula (IV).
(1) Taking 5kg of the green bamboo standard medicinal material, crushing, heating and refluxing with 95% ethanol at a solid-liquid ratio of 1:3 for three times of 2h, 1h and 1h respectively, combining the filtrates, carrying out reduced pressure rotary steaming, and freeze-drying to obtain 1kg of the green bamboo standard crude extract;
(2) adding appropriate amount of water into the crude extract, ultrasonic pulverizing, extracting with petroleum ether, ethyl acetate and n-butanol, filtering the extractive solution, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate, n-butanol and water extract;
(3) dissolving 180g of the obtained ethyl acetate part in methanol, adding 300g of 200-300-mesh silica gel for sample mixing, volatilizing the solvent, and performing volume ratio CH2Cl2Gradient elution with MeOH (100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 1:1 → 0:1) to give elution sites, concentration, 5000mL per gradient wash, one receiving volume per 500mL of receiving solution.
(4) Subjecting the first silica gel column chromatography CH2Cl2The first 2 receivers of the-MeOH (5:1) gradient elution site were combined and concentrated by C18Performing resin adsorption chromatography, and sequentially eluting with 12% MeOH, 30% MeOH, 45% MeOH, and 60% MeOH, wherein each gradient elutes 2500mL, each 500mL is 1 receiving solution, and the flow rate is 50 mL/min; c is to be18Passing the last 2 receiving volumes of the 45% MeOH elution part of the column chromatography through a Sepadex LH-20 gel resin chromatographic column, and eluting with MeOH, wherein each 10ml of the column chromatography is 1 receiving solution; subjecting the 6 th elution part of MeOH eluted from SephadexLH-20 gel column chromatography to CH3CN and H2Preparing a mobile phase with the volume ratio of O being 28:72, the flow rate being 3mL/min, the detection wavelength being 310nm, and injecting 50 muL each time to obtain the productCompound IV 35 mg.
Example 6
A process for the preparation of a compound of formula (IX).
(1) Taking 5kg of the green bamboo standard medicinal material, crushing, heating and refluxing with 95% ethanol at a solid-liquid ratio of 1:3 for three times of 2h, 1h and 1h respectively, combining the filtrates, carrying out reduced pressure rotary steaming, and freeze-drying to obtain 1kg of the green bamboo standard crude extract;
(2) adding appropriate amount of water into the crude extract, ultrasonic pulverizing, extracting with petroleum ether, ethyl acetate and n-butanol, filtering the extractive solution, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate, n-butanol and water extract;
(3) dissolving 180g of the obtained ethyl acetate part in methanol, adding 300g of 200-300-mesh silica gel for sample mixing, volatilizing the solvent, and performing volume ratio CH2Cl2Gradient elution with MeOH (100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 1:1 → 0:1) to give elution sites, concentration, washing 5000mL per gradient, one receiving volume per 500mL of receiving solution.
Subjecting the first silica gel column chromatography CH2Cl2The first 2 receiving volumes of the MeOH (5:1) gradient elution sites were pooled and concentrated by C18Performing resin adsorption chromatography, eluting with 12% MeOH, 30% MeOH, 45% MeOH, and 60% MeOH sequentially, wherein each gradient elutes 2500mL, each 500mL is 1 receiving volume, and the flow rate is 50 mL/min; c is to be18Passing the first 1 receiving solution of the 45% MeOH elution part of the column chromatography through a Sepadex LH-20 gel resin chromatographic column, and eluting with MeOH, wherein each 10mL is the receiving volume of 1 receiving solution; subjecting the 8 th elution part of MeOH eluted from SephadexLH-20 gel column chromatography to CH3CN and H2And preparing a mobile phase with the volume ratio of O being 28:72, wherein the flow rate is 3mL/min, the detection wavelength is 310nm, and 50 mu L of the compound IX is obtained by injecting each time.
And (3) structural identification: and (3) respectively measuring MS and NMR spectrums of the separated monomer components by using an Agilent 5973N mass spectrometer and a Burker 400MHz nuclear magnetic resonance spectrometer, wherein the obtained nuclear magnetic data are shown in a table 1-2, and identifying the structures of 6 new cyclotetetranol ester compounds IV, IX, V, VI, VII and VIII.
A compound IV: (2E,2' E) - (1R,2R,3S,4S) -3,4-dihydroxy-3- (3-methoxy-3-oxopropyl) cyclobutane-1,2-diylbis [3- (3,4-dihydroxyphenyl) acrylate]The chemical structure of the brown paste is characterized as shown in figures 1-2, and HR-ESIMS gives a molecular ion peak M/z531.8671[ M + H ]]+(calcd for C26H26O12531.1452), in combination1H NMR and13the C NMR spectrum of the compound IV presumes that the molecular formula is C26H26O12。13The C, HSQC and HMBC spectra show that the compound IV has 26 carbon atoms in total and comprises 1 methoxyl group (delta)C52.5), 2 methylene groups, 13 methine groups [ comprising 6 benzene ring carbons with chemical shifts δC115.3(C-2),116.3(C-5),121.8(C-6),115.2(C-2 "), 116.3 (C-5"), 121.9(C-6 "); 4 olefinic carbons, chemical shifts each deltaC146.0(C-7),114.3(C-8),146.2(C-7 "), 113.8 (C-8"); 3 continuous oxygen carbon with chemical shift deltaC72.5(C-1'),68.2(C-2'),65.8(C-4')]And 10 quaternary carbons [ including 2 ester carbonyl carbons conjugated to a double bond, each having a chemical shift of δC166.4(C-9) and δC165.7(C-9 "); 1 non-conjugated ester carbonyl carbon number deltaC173.8 (C-7'); 6 benzene ring carbons with chemical shifts of deltaC125.9(C-1),146.1(C-3),149.1(C-4),125.7(C-1 "), 146.1 (C-3"), 149.0(C-4 "); 1 to one oxygen carbon atom deltaC73.6(C-2')]。1H NMR spectrum shows 2 groups of caffeoyl signals: deltaH7.04(2H, overlapped, H-2,2 "), 6.76(2H, d, J ═ 8.0Hz, H-5, 5"), 6.99(2H, overlapped, H-6,6 "), and 4 trans olefinic hydrogen signals δH7.51(1H, d, J ═ 15.6Hz, H-7),6.26(1H, d, J ═ 15.6Hz, H-8),7.42(1H, d, J ═ 15.6Hz, H-7 "), 6.14(1H, d, J ═ 15.6Hz, H-8"), correlated by HMBC: h-7 and H-8/166.4 (C-9); this group is further demonstrated by H-7 "and H-8"/165.7 (C-9 "). Two ester carbonyl signals were found in the HMBC spectra to be related to two vicinal oxymethylene hydrogen signals, respectively: deltaH4.97(1H, dd, J ═ 2.4,6.0Hz, H-1') and 5.27(1H, d, J ═ 3.2Hz, H-2').1H 1H-1'/H-2' and H-1'/H-4' (delta. in the H-COSY spectrumH4.15) two sets of correlation signals indicate the presence of cafeoyl-O-C-1 ' H-C-2' H (O-cafeoyl) -C-3' (R)3And cafeoyl-O-C-1 'H-C-4' H (OR) -C-3'(R)3Coupling mode, together with the relevant signals in the HMBC spectra: h-1 'is related to C-2', C-3 'and C-4'; h-2 'is related to C-1', C-3 'and C-4'; h-4 'is related to C-1', C-2 'and C-3', demonstrating the presence of one cyclobutane group, and C-1 'and C-2' are linked to two caffeoyl ester carbonyl groups, respectively. In addition to this, the present invention is,1the H NMR spectrum also showed the presence of 1 methoxy group deltaH3.60(3H, s, OMe-7'), 4 high-field Hydrogen signals δH2.25(2H, overlaid, H-5'a, H-6' a),2.00(1H, D, J ═ 11.6Hz, H-5'b),1.89(1H, dd, J ═ 9.2,12.4Hz, H-6' b), by further 2D NMR analysis:1H 1H-COSY related signal H-5' a or H-6' a/H-5' b/H-6' b, HMBC related signal H-5' a or H-6' a is related to C-5', C-6', C-7', H-5' b is related to C-6', C-7', H-6' b is related to C-5', C-7', OMe-7' is related to C-7', and the existence of a methyl propionate group is proved. This group was shown to be attached to the C-3 'position of cyclobutane by correlation of H-5' a or H-6'a, H-5' b and H-6'b to C-3'. Finally, the C-3 'and C-4' linked groups of the cyclobutane groups were determined by 2D NMR and molecular formula analysis to be 2 OH: HMBC-related signal: OH-3' (delta)H5.91) associated with C-3' and C-5', OH-4' (delta)H5.17) related to C-1 'and C-4';1H 1H-COSY related signal: OH-4' (delta)H5.17) related to H-4'.
The relative configuration of the cyclobutane linking group is determined by NOESY correlation signals: the smaller ortho-coupling constants of H-1 'with H-2', H-4', H-5', H-6', H-2' with H-1', H-5', H-6', H-4' with H-1', H-5', H-6', and H-1' with H-2 'and H-1' with H-4 'determine that H-1', H-2', H-4', H-5', and H-6' are all in the beta configuration. The absolute configuration of compound iv was determined by a round two-chromatography method of fragmentation: comparing the CD spectra of the compounds IV and V shows that the CD curve of the IV has negative chirality, namely 345nm (delta epsilon-41.77) and 285nm (delta epsilon +20.70), so that two chromophores are arranged anticlockwise, thereby confirming that the C-2' of the compounds IV and V are both in R configuration, and further confirming the configuration of other chiral centers through NOE correlation. In summary, the structure of compound iv is identified as: (2E,2' E) - (1R,2R,3S,4S) -3,4-dihydroxy-3- (3-methoxy-3-oxopropyl) cyclobutane-1,2-diylbis [3- (3,4-dihydroxyphenyl) acrylate ].
A compound IX: (2E,2' E) - (1S,2S,3R,4R) -2,4-dihydroxy-2- (3-methoxy-3-oxopropyl)cyclobutane-1,3-diyl bis[3-(3,4-dihydroxyphenyl)acrylate]The chemical structure of the brown paste is characterized as shown in figures 3-4, and HR-ESIMS gives a molecular ion peak M/z529.0477[ M-H ]]-(calcd for C26H26O12529.1341), in combination1H NMR and13the C NMR spectrum assumed that Compound II has the formula C26H26O12And the molecular formula is consistent with that of the compound IV. Comparison of Compounds IX and IV1H NMR and13c NMR spectrum data show that the two groups are similar in structure, and the main difference is that the chemical shifts of C-1', C-2', C-3 'and C-4' of the cyclobutane groups are different, which shows that the positions of the substituent groups on the cyclobutane group in the compound IX are different from the positions of the substituent groups on the cyclobutane group in the compound IV. In the HMBC spectrum: h-1' (delta)H5.18) and C-9 (. delta.))C166.5),C-2'(δC71.4),C-3'(δC72.9),C-4'(δC67.1),C-5'(δC34.9) correlation; OH-2' (delta)H5.85) related to C-2 'and C-5'; h-3' (delta)H5.09) and C-9' (delta)C165.7),C-1'(δC70.4),C-2'(δC71.4) and C-4' (delta)C67.1) correlation; h-4' (delta)H3.87) and C-1' (delta)C70.4),C-2'(δC71.4),C-3'(δC72.9),C-5'(δC34.9) and C-6' (delta)C35.2) correlation; OH-4' (delta)H5.48) related to C-1 'and C-4'; h-5'a or H-6' a (. delta.) ofH2.20) and C-4' (delta)C67.1) correlation; h-5'b or H-6' b (. delta.) ofH2.20) and C-2' (delta)C71.4) correlation, and1H 1in the H-COSY spectrum: h-1 'is related to H-4'; h-3 'is related to H-4'; h-4 'is related to H-3'; OH-4' is related to H-4', indicating that two caffeate groups are respectively connected with C-1' and C-3' of cyclobutane, a methyl propionate group is connected with C-2', and two hydroxyl groups are respectively connected with C-2' and C-4 '. The relative configuration of the cyclobutane linking group is determined by NOESY correlation signals: the smaller ortho-coupling constants of H-1 'with H-2', H-4', H-5', H-6', H-2' with H-1', H-5', H-6', H-4' with H-1', H-5', H-6', and H-1' with H-2 'and H-1' with H-4 'determine that H-1', H-2', H-4', H-5', and H-6' are all in the beta configuration. Circular dichroism method for absolute configuration of compound IX by fragmentationDetermining: comparison of the CD spectra of compounds IX and IV revealed that the CD curves of IX exhibited negative chirality, 344nm (. DELTA.. di-elect cons. -7.02) and 285nm (. DELTA.. di-elect cons. +3.40), so that the two chromophores were arranged counterclockwise, thereby confirming that C-3' of compounds IX and IV were both of the R configuration, and the configuration of the other chiral centers was further confirmed by NOE correlation. In summary, the structure of compound ix was determined as: (2E,2' E) - (1S,2S,3R,4R) -2,4-dihydroxy-2- (3-methoxy-3-oxopropyl) cyclobutane-1,3-diylbis [3- (3,4-dihydroxyphenyl) acrylate]。
Compound v: (E) - (1R,2R,3S,4S) -2,3,4-trihydroxy-3- (3-methoxy-3-oxypropyl) cyclobutyl3- (3,4-dihydroxyphenyl) acrylate, brown paste, the chemical structure of which is characterized as shown in figures 5-6, HR-ESIMS (high-energy dispersive Mass Spectrometry) gives a molecular ion peak M/z367.0432[ M-H367.0432 ]]-(calcd for C17H20O9367.1024), in combination1H NMR and13the molecular formula of the compound V is presumed to be C by C NMR spectrum17H20O9。13The CNMR and HSQC, HMBC spectra showed that Compound V has a total of 17 carbon atoms, including 1 methoxy group (delta)C52.1), 2 methylene groups, 8 methine groups [ comprising 3 benzene ring carbons with chemical shifts δC115.2(C-2),116.3(C-5),121.7 (C-6); 2 olefinic carbons, chemical shifts each deltaC145.3(C-7),115.0 (C-8); 3 continuous oxygen carbon with chemical shift deltaC76.1(C-1'),64.9(C-2'),65.9(C-4')]And 6 quaternary carbons [ including 1 ester carbonyl carbon conjugated to a double bond, each having a chemical shift of δC166.7 (C-9); 1 non-conjugated ester carbonyl carbon number deltaC174.3 (C-7'); 3 benzene ring carbons with chemical shift deltaC126.1(C-1),146.1(C-3),148.8 (C-4); 1 to one oxygen carbon atom deltaC74.0(C-3')]. Comparison of Compounds V and IV1H NMR and13c NMR spectrum data show that the two structures are similar, and the main difference is that the compound V only contains one caffeate group, and the hydroxyl is substituted for the other caffeate group. This inference is further demonstrated by 2D NMR analysis, HMBC-related signal: h-1' (delta)H4.71) with C-9, C-2', C-3' (delta)C74.0), C-4 'and C-5' (delta)C38.4) correlation; h-2' (delta)H4.07) related to C-1', C-3' and C-4 '; OH-2' (delta)H4.90) withC-1 'and C-2' are related; OH-3' (delta)H5.67) related to C-3'; h-4' (delta)H3.95) related to C-1', C-2' and C-3 '; OH-4' (delta)H5.05) related to C-1 'and C-4'; h-5' a (. delta.) ofH2.08) and H-5' b (. delta.)H1.79) with C-2', C-4', C-6' (delta)C40.3),C-7'(δC174.3) correlation; h-6' a (. delta.) ofH1.87) and H-6' b (. delta.)H1.85) with C-3', C-5' (delta)C38.4), C-7';1H 1H-COSY related signal: h-1 'is related to H-4'; h-2' is related to H-4', H-5 '; h-4 'is related to H-1'; OH-2 'is related to H-2'; OH-4 'is related to H-4'. The relative configuration of the cyclobutane linking group is determined by NOESY correlation signals: h-1' is associated with H-2', H-4', H-5', H-6', H-2' is associated with H-1', H-5', H-6', H-4' is associated with H-1', H-5', H-6', and H-1' is associated with H-2' and H-1' is associated with H-4' with smaller ortho-coupling constants (J)H-1',2'2.8Hz and JH-1',4'6.8Hz) determined that H-1', H-2', H-4', H-5' and H-6' are all in the β configuration. The absolute configuration of the compound V is determined that C-2' of the compound V is R configuration by a split circular dichroism method, and the configuration of other chiral centers is further determined by NOE correlation. In conclusion, the structure of the compound V is determined as (E) - (1R,2R,3S,4S) -2,3,4-trihydroxy-3- (3-methoxy-3-oxypropyl) cyclobutyl3- (3,4-dihydroxyphenyl) acrylate.
Compound vi: (E) - (1S,2R,3R,4S) -2,3,4-trihydroxy-3- (3-methoxy-3-oxypropyl) cyclobutyl3- (3,4-dihydroxyphenyl) acrylate, brown paste, the chemical structure of which is characterized as shown in figures 7-8, HR-ESIMS gives a molecular ion peak M/z367.0440[ M-H367.0440 ]]-(calcd for C17H20O9367.1024), in combination1H NMR and13the C NMR spectrum of the compound IV presumes that the molecular formula is C17H20O9Corresponding to the molecular formula of the compound V. Comparing compounds VI and V1H NMR and13c NMR spectrum data show that the two groups have similar structures, and the main difference is that the chemical shifts of C-1', C-2', C-3 'and C-4' of the cyclobutane groups are different, which indicates that the position of the substituent on the cyclobutane group in the compound VI is different from that on the cyclobutane group in the compound V. In the HMBC spectrum: h-1' (delta)H5.00) and C-9 (. delta.))C165.8),C-2'(δC73.5),C-3'(δC67.4),C-4'(δC69.8),C-5'(δC35.6) correlation; h-2' (delta)H3.87) and C-1' (delta)C71.5), C-3', C-4' related; h-4' (delta)H3.57) related to C-1', C-2', C-3 '; h-5' a (. delta.) ofH2.10) and H-5' b (. delta.)H1.92) with C-2', C-4', C-6' (delta)C37.7),C-7'(δC174.1) correlation; h-6' a (. delta.) ofH2.10) and H-6' b (. delta.)H1.75) related to C-3', C-5', C-7', and1H 1in the H-COSY spectrum: h-1 'is related to H-4'; h-2' is related to H-4', H-5 '; h-4 'is related to H-1'; OH-2' (delta)H4.90) related to H-2'; OH-4' (delta)H5.05) associated with H-4', indicating that 1 caffeate group is linked to C-1' of cyclobutane, a methyl propionate group is linked to C-2', and three hydroxyl groups are linked to C-2', C-3 'and C-4', respectively. Since the NOESY association with the cyclobutane linking group is identical to compound v, the relative configurations are identical. The absolute configuration of the compound VI is determined that C-3' of the compound IV is R configuration by a split circular dichroism method, and the configurations of other chiral centers are further determined by NOE correlation. In conclusion, the structure of the compound VI is determined as (E) - (1S,2R,3R,4S) -2,3,4-trihydroxy-3- (3-methoxy-3-oxypropyl) cyclobutyl3- (3,4-dihydroxyphenyl) acrylate.
And (3) a compound VII: (E) - (1S,2R,3R,4S) -2- (3-ethoxy-3-oxopropyl) -2,3, 4-trihydroxybutyryl 3- (3,4-dihydroxyphenyl) acrylate, brown paste, the chemical structure of which is characterized as shown in figures 9-10, HR-ESIMS gives the molecular ion peak M/z381.0570[ M-H381.0570 ]]+(calcd for C18H22O9381.1180), in combination1H NMR and13c NMR spectra presume that the compound VII has the formula C18H22O9And a compound VI of the formula one more CH2. Comparing compounds VII and VI1H NMR and13and C NMR spectrum data show that the two structures are similar, and the main difference is that the methyl propionate group connected with C-2' in the compound VI is replaced by the ethyl propionate group. Correlation signals in HMBC spectra: h-5'a or H-6' a and H-5'b with C-2' (delta)C73.4),C-4'(δC69.4),C-6'(δC37.6),C-7'(δC173.5) related to each otherH-6'b and C-3' (delta)C67.3),C-5'(δC35.5), C-7' related, H2-1' with C-7', Me-2 ' (delta)C14.2) Me-2 "is associated with C-1", together with the relevant signals in the COSY spectra: h-5'a or H-6' a (. delta.) ofH2.09) with H-5' b (. delta.)H1.91) related, H-5' a or H-6' a/H-6' b (. delta.))H1.75) and H2-1”(δH3.94-4.07) and Me-2' (delta)H1.13) together with the above reasoning. Since the NOESY correlation and CD profile of the cyclobutane linking group is consistent with that of compound vi, both absolute configurations are consistent. In conclusion, the structure of the compound VII is determined as (E) - (1S,2R,3R,4S) -2- (3-ethoxy-3-oxopropyl) -2,3, 4-trihydroxybutyryl 3- (3,4-dihydroxyphenyl) acrylate.
Compound viii: (E) - (1S,2R,3R,4S) -2,3,4-trihydroxy-2- (3-methoxy-3-oxypropyl) cyclobutyl3- (4-hydroxypentyl) acrylate, brown paste, the chemical structure of which is characterized as shown in figure 11-12, HR-ESIMS gives a molecular ion peak M/z351.0502[ M-H ] M]+(calcd for C17H20O8351.1074), in combination1H NMR and13the C NMR spectrum of the compound VI presumes that the molecular formula is C17H20O8。13The CNMR and HSQC, HMBC spectra show that Compound VIII has a total of 17 carbon atoms, including 1 methoxy group (delta)C52.1), 2 methylene groups, 9 methine groups [ comprising 4 benzene ring carbons with chemical shifts δC130.7(C-2, C-6),116.3(C-3, C-5); 2 olefinic carbons, chemical shifts each deltaC145.2(C-7),114.6 (C-8); 3 continuous oxygen carbon with chemical shift deltaC71.5(C-1'),67.5(C-3'),70.0(C-4')]And 5 quaternary carbons [ including 1 ester carbonyl carbon conjugated to a double bond, each having a chemical shift of δC165.9 (C-9); 1 non-conjugated ester carbonyl carbon number deltaC174.1 (C-7'); 2 benzene ring carbons with chemical shift of deltaC125.4(C-1),160.4 (C-4); 1 to one oxygen carbon atom deltaC73.6(C-2')]. Comparing compounds VIII and VI1H NMR and13c NMR spectrum data show that the two structures are similar, and the main difference is that the caffeic ester group in the compound VI is replaced by p-hydroxycinnamic acid ester group. Interpretation of the 1D and 2D NMR data further confirms the above inference:1an AB coupling system exists in H NMR spectrum, and hydrogen signals are respectively deltaH7.53(2H, d, J ═ 8.4Hz, H-2, H-6), 6.81(2H, d, J ═ 8.4Hz, H-3, H-5); correlation signals in HMBC spectra: h-2 and H-6 with C-1 (. delta.)C125.4),C-3,C-5,C-4(δC160.4),C-7(δC145.2), H-3 and H-5 are related to C-1, C-2, C-4, C-6, together with the related signals in the COSY spectra: h-2 and H-6 are related to H-3 and H-5. Since the NOESY correlation and CD profile of the cyclobutane linking group is consistent with that of compound vi, both absolute configurations are consistent. As described above, the structure of compound VIII is defined as (E) - (1S,2R,3R,4S) -2,3,4-trihydroxy-2- (3-methoxy-3-oxypropyl) cyclobutyl3- (4-hydroxyphenyl) acrylate.
TABLE 1 of Compounds V to IX1H NMR spectroscopic data (400MHz, DMSO-d)6,δppm,J,Hz)
Figure GDA0003308701230000161
TABLE 2 of compounds V to IX13C NMR spectroscopic data (400MHz, DMSO-d)6,δppm)
Figure GDA0003308701230000162
Figure GDA0003308701230000171
Pharmacological experiment:
1. experimental Material
Reagents and instrumentation: the compounds V to IX are obtained by separating the subject from the green bamboo marks; DPPH (Sigma, usa); multifunctional microplate reader (Tecan, France).
2. Test method
And (3) measuring antioxidant activity:
DPPH free radical scavenging experiments: adding 50 μ L of sample into 96-well plate, adding 100 μ L of freshly prepared DPPH methanol solution, mixing, shaking at 200r/min for 1min, incubating at room temperature in darkIncubate for 30min, set 5 concentrations for the sample, 90,60,30,15,7.5ppm respectively. A DPPH blank and a methanol blank were set simultaneously, wherein the DPPH blank changed a 50. mu.L sample to 50. mu.L methanol and the methanol blank added 150. mu.L methanol. Measuring OD value of each compound at wavelength of 517nm with microplate reader, repeating the measurement for 3 times, and calculating corresponding inhibition rate and IC50The value is obtained. The formula for calculating the clearance rate of free radicals is as follows: IR (%) ═ aDPPH-Atest)/(ADPPH-AMeOH) Wherein A isDPPHIs the OD value of DPPH blank group, AtestOD value of sample group, AMeOHThe OD value is the blank group of methanol.
3. Results of the experiment
From the above experimental results, it can be seen that compound iv has strong DPPH radical scavenging activity, i.e., antioxidant activity, and at a concentration of 100ppm, the radical scavenging rate to DPPH is 94.04%, and that compounds ix, v, vi, vii, viii have weak antioxidant activity, and the radical scavenging rates to DPPH are 24.1%, 17.5%, 14.6%, 9.8% and 8.9%, respectively.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (25)

1. A method for extracting cyclotetetranol ester compounds is characterized by comprising the following steps: the method comprises the following specific steps:
performing crude extraction on the green bamboo label, sequentially extracting the crude extract by using petroleum ether and ethyl acetate, and removing a solvent from an extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
the ethyl acetate extract was dissolved in CH2Cl2-MeOH, gradient elution with silica gel chromatography, gradient elution of CH2Cl2-MeOH in the sequence 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1, dividing the eluent of each gradient into 10 portions of receiving solution;
combining the receiving solutions of different segments according to the receiving sequence, concentrating, and passing the concentrate through C18Elution with an adsorbent resin column, C18Gradient eluents of the adsorption resin chromatographic column are 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH in sequence, and each gradient eluent is divided into 5 parts of receiving solution;
taking different sections of receiving liquid according to the receiving sequence, and using CH3CN and H2Eluting and separating the mixed solution of O to obtain a compound shown in the formula (I);
the structure of the compound shown in the formula I is as follows:
Figure FDA0003308701220000011
wherein R is1Is selected from
Figure FDA0003308701220000012
R2Is selected from
Figure FDA0003308701220000013
Or a hydrogen radical; r3Selected from hydroxy or
Figure FDA0003308701220000014
R4Selected from hydrogen radicals or hydroxyl radicals.
2. The method for extracting cyclotetritol ester compounds as claimed in claim 1, wherein: in the first gradient elution with silica gel chromatographic column, the first 5 receiving solutions with gradient of 15:1 are combined and concentrated to obtain concentrate A, which is dissolved in CH2Cl2-MeOH in a volume ratio of 15:1, and gradient elution with CH by silica gel chromatography2Cl2-MeOH at a volumetric ratio of 100:1 → 50:1 → 25:1 → 15:1, dividing the eluate from each gradient into 10 portions of the receiving solution, and combining the receiving solutions from the 15:1 gradients to obtain an eluted fraction B;
elution fraction B through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and dividing each gradient eluent into 5 parts of receiving solution;
the 5 th receiver of the elution fraction of a 30. + -. 1% MeOH gradient was treated with CH3CN and H2Taking the mixed solution of O as a mobile phase for elution and separation to obtain a compound shown in a formula (VI);
the compound of formula VI has the structure:
Figure FDA0003308701220000021
3. the method for extracting cyclotetritol ester compounds as claimed in claim 2, wherein: CH (CH)3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 15: 80-90.
4. The method for extracting cyclotetritol ester compounds as claimed in claim 2, wherein: elution was with MeOH, which flowed at 45-55 mL/min.
5. The method for extracting cyclotetritol ester compounds as claimed in claim 2, wherein: by CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
6. The method for extracting cyclotetritol ester compounds as claimed in claim 1, wherein: in the first silica gel column gradient elution, the first 5 receiving solutions with gradient of 15:1 are combined and concentrated to obtain concentrate C, and the concentrate C is dissolved in CH2Cl2-MeOH in a volume ratio of 15:1, and gradient elution with CH by silica gel chromatography2Cl2MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1, dividing each gradient into 10 parts of the receiving solution, and combining the receiving solutions of 15:1 gradient to obtain the eluted fractionD；
Elution fraction D through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and taking 5 parts of receiving solution as eluent of each gradient;
the 3 rd receiver of the elution fraction with a 45. + -. 1% MeOH gradient was applied with CH3CN and H2Taking the mixed solution of O as a mobile phase for elution and separation to obtain a compound shown as a formula (VII);
the structure of the compound shown in the formula VII is as follows:
Figure FDA0003308701220000022
7. the method for extracting cyclotetritol ester compounds as claimed in claim 6, wherein: CH (CH)3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 20: 75-95.
8. The method for extracting cyclotetritol ester compounds as claimed in claim 6, wherein: elution was with MeOH, which flowed at 45-55 mL/min.
9. The method for extracting cyclotetritol ester compounds as claimed in claim 6, wherein: by CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
10. The method for extracting cyclotetritol ester compounds as claimed in claim 1, wherein: performing gradient elution with silica gel chromatographic column for the first time, concentrating the last 5 receiving solutions with gradient of 15:1 to obtain concentrate E, and dissolving concentrate E in CH2Cl2-MeOH in a volume ratio of 15:1, and gradient elution with CH by silica gel chromatography2Cl2MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1, run each gradient of eluentDividing the receiving solution into 10 parts, and combining the receiving solutions with a gradient of 15:1 to obtain an elution part F;
elution fraction F through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and dividing each gradient eluent into 5 parts of receiving solution;
the 2 nd receiver of the elution fraction of a 30. + -. 1% MeOH gradient was treated with CH3CN and H2Taking the mixed solution of O as a mobile phase for elution and separation to obtain a compound shown in a formula (V);
the structure of the compound shown in the formula V is as follows:
Figure FDA0003308701220000031
11. the method for extracting cyclotetritol ester compounds as claimed in claim 10, wherein: CH (CH)3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 14: 80-90.
12. The method for extracting cyclotetritol ester compounds as claimed in claim 10, wherein: elution was with MeOH, which flowed at 45-55 mL/min.
13. The method for extracting cyclotetritol ester compounds as claimed in claim 10, wherein: by CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
14. The method for extracting cyclotetritol ester compounds as claimed in claim 1, wherein: in the first silica gel chromatographic column gradient elution, the last five receiving solutions with the gradient of 10:1 are merged and concentrated to obtain a concentrate G; passing the condensate G through C18Sequentially eluting with 12 + -1% MeOH, 30 + -1% MeOH, 45 + -1% MeOH, and 60 + -1% MeOH, and separating the eluates of each gradient5 parts of receiving solution; passing the second 2 receiving solutions of the 45 +/-1% MeOH gradient elution part through a Sepadex LH-20 gel resin chromatographic column, and eluting with MeOH, wherein the eluent is divided into more than 8 receiving solutions;
elution fraction 3 with MeOH site CH3CN and H2Taking the mixed solution of O as a mobile phase for elution and separation to obtain a compound shown as a formula (VIII);
the structure of the compound shown in the formula VIII is as follows:
Figure FDA0003308701220000032
15. the method for extracting cyclotetritol ester compound as claimed in claim 14, wherein: CH (CH)3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 20: 75-85.
16. The method for extracting cyclotetritol ester compound as claimed in claim 14, wherein: elution was with MeOH, which flowed at 45-55 mL/min.
17. The method for extracting cyclotetritol ester compound as claimed in claim 14, wherein: by CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
18. The method for extracting cyclotetritol ester compounds as claimed in claim 1, wherein: in the first silica gel chromatographic column gradient elution, the first 2 receiving solutions with a gradient of 5:1 are combined and concentrated to obtain a concentrate H; passing concentrate H through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and dividing each gradient eluent into 5 parts of receiving solution; the second 2 receivers of the 45. + -. 1% MeOH gradient elution fraction were passed through a Sephadex LH-20 gel resin column and run with MeOHEluting, wherein the eluent is divided into more than 8 parts of receiving solution;
elution fraction 6 with MeOH site CH3CN and H2Taking the mixed solution of O as a mobile phase for elution and separation to obtain a compound shown in a formula (IV);
the structure of the compound shown in the formula IV is as follows:
Figure FDA0003308701220000041
19. the method for extracting cyclotetritol ester compound as claimed in claim 18, wherein: CH (CH)3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 28: 70-75.
20. The method for extracting cyclotetritol ester compound as claimed in claim 18, wherein: elution was with MeOH, which flowed at 45-55 mL/min.
21. The method for extracting cyclotetritol ester compound as claimed in claim 18, wherein: by CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
22. The method for extracting cyclotetritol ester compounds as claimed in claim 1, wherein: in the first silica gel chromatographic column gradient elution, the first 2 receiving solutions with a gradient of 5:1 are merged and concentrated to obtain a concentrate K; through C18Sequentially eluting the adsorption resin chromatographic column with 12 +/-1% of MeOH, 30 +/-1% of MeOH, 45 +/-1% of MeOH and 60 +/-1% of MeOH, and dividing each gradient eluent into 10 parts of receiving solution; passing the first 1 receiving volumes of the second part of the 45 +/-1% MeOH gradient elution part through a Sepadex LH-20 gel resin chromatographic column, eluting with MeOH, and dividing the eluent into more than 8 parts of receiving solution;
elution fraction 8 with MeOH with CH3CN and H2Eluting and separating the mixed solution of O as a mobile phase to obtain a compound shown as a formula (IX);
the structure of the compound shown in the formula IX is as follows:
Figure FDA0003308701220000051
23. the method for extracting cyclotetritol ester compound as claimed in claim 22, wherein: CH (CH)3CN and H2In a mixed solution of O, CH3CN and H2The volume ratio of O is 28: 70-75.
24. The method for extracting cyclotetritol ester compound as claimed in claim 22, wherein: elution was with MeOH, which flowed at 45-55 mL/min.
25. The method for extracting cyclotetritol ester compound as claimed in claim 22, wherein: by CH3CN and H2And taking the mixed solution of O as a mobile phase for elution and separation, wherein the flow rate of the mobile phase is 2-4 mL/min.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004000302A1 (en) * 2002-06-25 2003-12-31 Raju Gokaraju Ganga Novel resveratrol analogs
CN102850220A (en) * 2012-10-11 2013-01-02 南京农业大学 Sea asparagus ferulic acid ester compound as well as preparation method and application of compound
CN105367613A (en) * 2015-12-16 2016-03-02 海南大学 Preparation method of antioxidant acteoside in Clerodendron cyrtophyllum
CN107325140A (en) * 2017-07-21 2017-11-07 山东省分析测试中心 A kind of chromone ketoside compounds isolated and purified from blue or green bamboo mark and extracting method
CN107698510A (en) * 2017-10-27 2018-02-16 山东省分析测试中心 The alkaloid compound and extracting method extracted from blue or green bamboo mark

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004000302A1 (en) * 2002-06-25 2003-12-31 Raju Gokaraju Ganga Novel resveratrol analogs
CN102850220A (en) * 2012-10-11 2013-01-02 南京农业大学 Sea asparagus ferulic acid ester compound as well as preparation method and application of compound
CN105367613A (en) * 2015-12-16 2016-03-02 海南大学 Preparation method of antioxidant acteoside in Clerodendron cyrtophyllum
CN107325140A (en) * 2017-07-21 2017-11-07 山东省分析测试中心 A kind of chromone ketoside compounds isolated and purified from blue or green bamboo mark and extracting method
CN107698510A (en) * 2017-10-27 2018-02-16 山东省分析测试中心 The alkaloid compound and extracting method extracted from blue or green bamboo mark

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Chemical Constituents from Scindapsus officinalis (Roxb.) Schott. and Their Anti-Inflammatory Activities;Hongjing Dong et al.;《MOLECULES》;20180831;第23卷(第10期);第1-10页 *
Isolation and Structure Elucidation of Secondary Metabolites from the Roots of the Tunisian Convolvulus dorycnium;Manel Hassine et al.;《CHEMISTRY OF NATURAL COMPOUNDS》;20160930;第52卷(第5期);第830-833页 *
Phenolic constituents of Convolvulus dorycnium L. flowers;Sana Nacef et al.;《Phytochemistry Letters》;20100611;第3卷(第2期);第66-69页 *

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